Various chemical constituents of DNA extraction buffer allow effective isolation. 5. DNA Purification | DNA Extraction Methods | Promega - Promega Corporation May need to be heated to 50C to dissolve. We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. Tannin is actually the main culprit. Discard all waste phenol and chloroform into chemical waste bottle in fume hood. iKjA3)])"ZcaR*.&p)n5x.9,Vjt_* K;lZe0# ;Iv1WUbg>s{x,4L4(@[xidmy)dK4^Cv$[1` 8[O16Gok9$pZBQM'!(H0RK9ra)"}Vjo7`/J1j#3g3]( eo; M(]+d[` mAQ8 rk-%wURl0]vcRBqR^W(1+.Ho)T-^gbrGvVp-{x%`1wE;b,uD^Y{_o Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Do not sell or share my personal information, 1. Free access to premium services like Tuneln, Mubi and more. Plant DNA extraction is a hard, tedious and time-consuming process. The mantra to success in plant DNA extraction is to grind tissue well, hard, and rough until fine homogenization. I will also explain variations and concentrations of different CTAB buffers that you can try and use. Add liquid nitrogen to the tissue and roughly grind the sample into the mortar and pestle. KIT METHOD OF DNA EXTRACTION. Following plant cell lysis, RNase A is added to digest RNA, and DNA is separated from other cellular components using phenol/chloroform extraction, which separates the sample into two distinct aqueous and organic phases after centrifugation. First report of 'Candidatus Phytoplasma australasia' related strain overview before delving deeper into the procedure in a step-by-step approach. The basic principle of DNA isolation is disruption of the cell wall, cell membrane, and nuclear membrane to release the highly intact DNA into solution followed by precipitation of DNA and removal of the contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols, and other secondary metabolites by enzymatic or chemical For the extraction and purification of DNA from leaves and seeds, cetyltrimethylammonium bromide (CTAB) is the "go-to" procedure. Purvi Jain - Perfumer - Sawai Fragrances | LinkedIn DNA Extraction - Genomics Corpus ID: 102206113; Comparison of Four Methods of DNA Extraction from Needles of Pinus tabulaeformis @article{Ming2012ComparisonOF, title={Comparison of Four Methods of DNA Extraction from Needles of Pinus tabulaeformis}, author={Li Ming}, journal={Hubei Agricultural Sciences}, year={2012} } You have to modify the protocol and preparation as per your need. ii. Get news, product info, tips, industry updates, events, freebies, and more delivered right to your inbox. The method is fast,. CTAB buffer (preheated at 65C for 15 min.) startxref 4. This is because the base pairs in DNA absorb UV light, therefore amount of bp is directly proportional to DNA concentration. (CTAB) contained in the extraction buffer. Under these Dellaporta (1983) and Maroof et al., (1984) have optimized the existing protocols and developed excellent methods for plant DNA extraction. and nucleus wall. The first, and now the most numerous, organisms isolated from deep-sea hydrothermal vents belong to the order Thermococcales, within the Euryarchaeota in the domain Archaea. For this Now add chilled isopropanol (70%), and a pinch of NaCl to the supernatant and precipitate the DNA by inverting the tube several times. PDF Download Free Modern Biology Section 17 Section Pdf The structure is explained in the figure below. The structure and concentration of these compounds can also vary substantially between different plant species, making the development and optimization of a one size fits all CTAB protocol difficult.8,9 In addition, phenol and other salts introduced throughout your CTAB protocol can remain, even after extensive ethanol washes. We've updated our privacy policy. Tap here to review the details. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added. In 1980, Murray and Thompson proposed an inexpensive and simple protocol for plant DNA extraction by CTAB. Looks like youve clipped this slide to already. Take 5gms of fresh plant tissue and cut it into small pieces. A total of 30 samples of five algal genus were used in this study. Phenol is toxic and corrosive to skin and eyes. of Red Blood Cells, RBC. Nederlnsk - Frysk (Visser W.), Applied Statistics and Probability for Engineers (Douglas C. Montgomery; George C. Runger), How to extract plant DNA using CTAB method, 7 Chap 11 (Bioenergetic) F.Sc 1st Year Biology Helping Notes, L.N.Gumilyov Eurasian National University, Kwame Nkrumah University of Science and Technology, Jomo Kenyatta University of Agriculture and Technology, Constitutional law of Ghana and its history (FLAW306), Probability and statistics for engineers (MKT3802), Business Communication & Interpersonal Skills (MCM 509), Avar Kamps,Makine Mhendislii (46000), Power distribution and utilization (EE-312), Chapter 03 - The Time Value of Money (Part 1), SMA 2231 Probability and Statistics III course outline. We use cookies to track visits to our website, and we don't store any of your personal details. Enjoy access to millions of ebooks, audiobooks, magazines, and more from Scribd. 2023 Zymo Research Corporation. PDF Bacterial genomic DNA isolation using CTAB - DOE Joint Genome Institute Phenol, chloroform and isoamyl alcohol DNA extraction. Dissolve in 50 L stream For example, some plants need only simple grinding in mortar and pestle, while some need liquid nitrogen. DNA Extraction - Cells are resuspended in 0.8 mL of pre-warmed (60C) CTAB extraction buffer CTAB buffer 2% CTAB (hexadecyltrimethylammonium bromide) 100 mM TrisHCl [pH=8] 20 mM EDTA, 0.2% -mercaptoethanol [added just before use] 1.4 M NaCl 0.1 mg/mL proteinase K [added just before use]) MilliQ or Nanopure) and filter sterilize (push solution INORGANIC METHOD OF DNA EXTRACTION 3. trailer DNA extraction by chromatography: 2. homogenate to sterile Eppendorf tube. PPT PowerPoint Presentation Cell preparation and extraction techniques. additional purification, or wash, of the nucleic acid from the remaining salt. If you will be using it a lot, consider making multiple smaller aliquots, since repeated freeze/thawing will deteriorate the quality. The solid phase protocol listed below is an alternative. The precipitate is pelleted by centrifugation and washed with 70% ethanol to remove salts introduced during extraction. Use 70% ethanol to wash DNA pellet. A General DNA extraction scheme includes cell lysis, removal of contaminant and DNA stabilizing, precipitation and elution steps. There are also more than 20 steps in the protocol and as the number of samples increases, the amount of time needed to complete DNA extraction increases substantially. Rapid isolation of high molecular weight plant DNA. Plant DNA extraction - CTAB Method - YouTube Ethanol and NaCl are used to remove plants polysaccharides. Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however the fundamentals of DNA extraction remains the same. PowerPoint: Audio and Video Product Design: From Sketch to CAD . The hazard with traditional CTAB protocols is the protein component of plant lysates is usually removed using phenol and chloroform. Maria Kotsaridi - Laboratory Administrator of molecular genetics Add 500 l chloroform, vortex, spin 5 min, retain supernatant. To keep up with this rapidly advancing field, DNA extraction protocols must be robust, flexible, consistent, and fast. buffering capacity (a low or high pH damages DNA). However, the CTAB-like buffer system can do better with such hard tissues. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[250,250],'geneticeducation_co_in-box-4','ezslot_21',149,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-box-4-0'); Go through this article: Why is DNA extraction from plants difficult? out, while the nucleic acid precipitates. Kamber T, Malpica-Lpez N, Messmer MM, et al. This will increase the yield. Proteinase K DNA extraction: 6. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are often included in protocols for DNA . In this paper, most frequently used techniques for DNA extraction from insect with different size and condition are outlined. Magnesium is a cofactor for DNase. Washing and elution- By alcohol and TE (Tris-EDTA) buffer. QLnoSc3 TuAWbAJ7$ (5hiHFn%wCEJ}=PbcSB#hN(YYXl69Dr ]\79/SiSM J,QOY5#^A@r|)(' g@9_d[v9|L.Th*9I5D(Y Follow this protocol on how to use PCI: Phenol, chloroform and isoamyl alcohol DNA extraction. Extraction of DNA, RNA, and protein is the basic method used in molecular biology. 0000000812 00000 n By accepting, you agree to the updated privacy policy. The distribution of alpine herbs is severely threatened by climate change, which influences genetic variation and population structure. x$)FcD2k@ c@5#3Q`e^KDzqjpcm|;lm+ou>oyu?mei{:.||vtz~mG18zh]w \v?o.?;]wWv:auaw:t@-Og8ArNg(quz|JFv\'nC~w^P2uwpvRwghEPC"w;x@wb:^Q$~P9&*RlJAWfb:wT]8PJ\*u;Tut7.TJOD$F:ZTi/=l6QMoT 7j!Fq6bIXuO07:Ro2St~Ovt4',b6bJQ\v:"E 0000001496 00000 n method, providing an overview before delving deeper into the procedure in a step-by-step approach. A lysozyme incubation can be added if cells dont lyse well with CTAB alone. DNA extraction is a process of isolating DNA from any biological cell to use it for analysis like PCR, sequencing or restriction digestion. Take a sample in the Eppendorf tube and centrifuge it at 12,000rpm for 2 minutes. The molecular formula of CTAB is [(C16H33)N(CH3)3]Br. Other methods of DNA extraction include salting out , cesium chloride density gradients , and chelex 100 resin [16, 17]. One in, one out: Generic circumscription within subtribe Manilkarinae We've updated our privacy policy. UV absorbance spectrometry. uT`_zYduD=k isj`|p[!jhP9v7(v4x[P9 *n Mu^265!/f^P lNto5Yzs$xZ:4Qu.|fS=L;8cwX0 extraction and purification of DNA from plants and plant derived foodstuff and is particularly Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.). In this case, after adding TE and NaCl to cell pellet, add lysozyme to 1 mg/ml, and incubate 1 hour at 37C. The DNA from leaf samples of agarwood-producing plants was isolated using the CTAB method (Gholibeigian 2021) The matK, ITS, and trnL-trnF gene fragments were sequenced and used to confirm the . Institute of Biomedical and Genetic Engineering (IBGE), Dna fingerprinting matreilas & methods of chilli, Dna extraction from fresh or frozen tissues, Human biosafety issues of nanomaterials used as nanomedicine, Introduction, pathophysiology and treatment options for covid 19, Inhibition of Gene Expression at RNA Level. Inorganic DNA Extraction: 6M . After the tissue becomes a powder, add 500 L of CTAB extraction buffer and beta-mercaptoethanol, grind it again followed by vortexing for 5 minutes. EXTRACTION (i) Grind 2 to 5 g of frozen leaves to a very fine powder using N2 cooled mortar and pestle. | DNA Extraction Method 2. Academia.edu no longer supports Internet Explorer. "Processing with GMO detection method and confirmation of results for corn, soy, cotton and tomato."-Microorganisms' cultivation.-DNA extraction (CTAB ptotocol) -Method confirmation according to ISO 17025.-PCR (Polymerase chain reaction) . PDF Plant Genomic DNA Extraction by CTAB 2 Fiona - University of Queensland Clipping is a handy way to collect important slides you want to go back to later. PDF DNA Extraction and Gel Electrophoresis INTRODUCTION (ii) Add 25 ml CTAB buffer and transfer to 50 ml tube. Gw.:S /vR.fP" pbgQ$C e;=AE-0I's>rEwd z.Ar1-tAMF.~V\2;MC#:- 54o)IR pZ|h!0"# x\KsFW1XD$I~,5>x,}HeVVwmk-m?X/VW|\ It is imperative that sufficient cells are collected. Isolation of good quality DNA is a fundamental first step in insect DNA barcoding which is obtained by standardizing the DNA isolation method. Alex: Gee, thats a lot of chloroform, might be a while before I risk trying this protocol. Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. mix gently by inverting and spin at 13,000 rpm at 4C for 10 min. v;K6n8>0xx~fzNFLscB=)lo-s7iW:QxzA+ o45bW`=bN htt& All Rights Reserved. The use of Urea is the easy and safest way to extract DNA from plants and sufficient enough for routine PCR and restriction digestion but not enough for sequencing or microarray analysis. 10 Different Types of DNA Extraction Methods (Updated) - Genetic Education Heating at 50-60C (up to an hour, intermittent mixing) and/or addition of more EB may be required to dissolve all the DNA. 0000000893 00000 n Techniques of DNA Extraction, Purification and Quantification, Nucleic Acid Quantification Methods - DNA / RNA Quantification, Isolation and purification of microbial c, DNA- Basics on isolation, quantification, storage, Application of molecular technology in biotechnology, Sarhad University of Science and Technology, Biotechnology experiments 2nd semester (LNMU Darbhanga), International Medicine School - Management and Science University, Dna extraction strawberry lab spring 2015, Extraction buffer, Protease inhibitors methods of cell distrubtion, KYBELLA AND OTHER FILLERS USED FOR BEAUTY ENHANCEMENT. 4UYG$(. How to Extract DNA From Plant Leaves & Seeds | ZYMO RESEARCH H'8R(|Zp{7OeCl _Y&!S5sUFF$>`! q;>:V^6FUW>!Aq2WF 10 Secret Tips for DNA Extraction to Get Good Results. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Steps in Organic and Inorganic DNA Extraction. Lysis of cell membrane: The first step of the DNA extraction is the rupture of the cell Protocol for Genomic DNA extraction with CTAB NeoSynBio Clipping is a handy way to collect important slides you want to go back to later. CTAB Method Abbreviated: Cetyl trimethylammonium bromide Function: CTAB buffer is used to lyse plant cells in the same way SDS is used to lyse bacterial cells. However, in practice, the protocol has several drawbacks: its lengthy, tedious, and low throughput, with many steps that require careful handling, exposure to hazardous chemicals, and several other technical considerations. Research . Saturated NaCl was needed for the proteins to precipitate out of the solution. Plant cell walls are very difficult to break down and the cells contain many compounds that impede extraction and inhibit downstream molecular biology applications. Most of the time students fail to extract good DNA. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. the DNA purity and therefore quality. Safe Use of Phenol | Safety Services. Notes from Kazakh Khanate till Political Dynamics + reading answers, Solutions - 6th Edition Computer and Networking, Stock Watson 3U Exercise Solutions Chapter 5 Instructors, A sample of letter of enumerator addressed to your employer, The Love Hypothesis Chapter 16 Adams POV by Ali Hazelwood (z-lib, Business Statistics and Mathematics Solved Past Papers, B.com Part 1 Punjab University 2009-2018, Siki soku 104 - dg sdg sdg fdg dsf sdg sdg sdg fdg dsf sdg sdg sdg fdg dsf sdg sdg sdg fdg dsf. A qPCR Assay for the Fast Detection and Quantification of Colletotrichum lupini. This research evaluated five genome extraction methods including Protocol A (main CTAB), Protocol B (CTAB without -mercaptoethanol), Protocol C (CTAB without ammonium acetate), Protocol D (modified method of Murray and Thompson), and Protocol E (Gene All-Kit), to determine their ability to extract DNA from fresh and herbarium leaves of A. fridae. Angeles JGC, Laurena AC, Tecson-Mendoza EM. The Cetyl Trimethyl Ammonium A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. By accepting, you agree to the updated privacy policy. However, care must be taken while handling liquid nitrogen. After grinding, the tissue is transferred to a tube and CTAB buffer is added. 0000002963 00000 n % K6-0001-050 or -200) is designed for processed and/or multi-ingredient foods. Quality analysis of genomic DNA and authentication of fisheries PDF DNA Extraction From Blood - KSU A method for DNA Extraction and molecular identification of Aphids. Plant genomic DNA extraction - YouTube of 0 TE+ RNase (100 g/ml). However, the use of a CTAB buffer will surely help in the majority of cases. Thus even if your DNA is good and your PCR fails repeatedly, you would have to worry about tannin. A modified protocol for rapid DNA isolation from plant tissues using Extraction of high-quality genomic DNA from different plant orders In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less Once the ethanol is decanted, the residual ethanol in the pellet is evaporated away and the dried pellet is resuspended in your buffer-of-choice for your downstream application, such as PCR or NGS. Magnetic bead-based DNA extraction: 8. The evaluated items included DNA . It was developed in the 1980s and has been used ever since, with various modifications for different plant species.2-5. >,xPD/-}V62h (s['HXGF=_fCUVY~ .ET_Xwk6A.xYkNMaR^+C?x'"LhaxDU|&O'Z Effects of symbiotic population impairment on microbiome composition Vortexing steps can be replaced or supplemented by inversion and flicking of tube. Spin-column DNA extraction: 7. It instantly froze tissue below its freezing temperature and eventually ruptures the cell wall. Click here to review the details. The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing, and put into a solution containing salt. Its better to leave a trace of ethanol and water in the pellet (and have it dissolve easily), than to remove all moisture, and then have great difficulty dissolving the pellet. Incubate 1 hr at 37C. 0 The procedure for fixing the aptamer onto these transducers and for monitoring the interaction with the target protein is shown here. 2. You choose the most appropriate method ! Use of phenol/chloroform also generates organic waste which requires special storage containers and disposal procedures. This plasmid, pGT5, is a stable multicopy plasmid (2530 copies per chromosome) and can be isolated from cultures at different growth phase and in the temperature range from 75 up to 105oC. Take 100 mg tissue, homogenate PDF Principle of DNA Extraction - kau x0pnzmM )3jo.o8e'nn/j!;+RRRJ8F9k^ iN Qfe=\%A9BL YLoa 8C c)o63Z1`*4#q U0 After the clear pellet appears, add 500L of TE buffer or elution buffer to the pellet and dissolve the DNA in it. Bring down the sample temperature to RT, add 600L chloroform: isoamyl alcohol (24:1), Results indicated that out of five extraction methods, the use of SDS resulted to the highest quality and quantity of DNA, followed by the CTAB method. PROJECT ON WASTE WATER TREATMENT AND ENERGY PRODUCTION FROM SLUDGE AND SOLID FERMENTATION: TECHNIQUE USED FOR THE PRODUCTION OF FOOD BY MICROBES, Human glucose-6-phosphate isomerase(GPI) or phosphoglucose isomerase(PGI), PHOSPHOLIPIDS, GLYCEROPHOSPHOLIPIDS, SPHINGOMYELIN AND GLYCOLIPIDS, Simulations of Test Reduction Using Pooled Heavy Metals Analysis inCannabis, Quantized space time and Einstein's special theory of relativity.pdf. There are two reasons for that. Our binding chemistry, wash solutions, and spin column technology remove polysaccharides, lipids, and other common downstream inhibitors and contaminants providing ultra-pure DNA, with minimal loss. Rl%= Aboul-Maaty NAF, Oraby HAS. Precipitation Part I: phenol/chloroform extraction to get rid of proteins. ORGANIC METHOD OF DNA EXTRACTION 2. Centrifuge the sample at 25,000rpm for 5 to 8 minutes and transfer the supernatant into another tube. 0000003202 00000 n Towards DNA-Based Methods Analysis for Honey: An Update In addition, the presence of turgor pressure, all these things creates problems in plant DNA extraction. They work in a similar fashion, apart from CTAB being a cationic detergent while SDS is anionic. )Z$m|h_8"dOtv#P%w;:K&z]x.A`5.AZ%q9;eU! endstream endobj 38 0 obj <> endobj 39 0 obj <>/Font<>/ProcSet[/PDF/Text]>>/Rotate 0/Type/Page>> endobj 40 0 obj <> endobj 41 0 obj <> endobj 42 0 obj <> endobj 43 0 obj <> endobj 44 0 obj <> endobj 45 0 obj <> endobj 46 0 obj <> endobj 47 0 obj <>stream First, they dont know which buffer to use and second, they even dont know how to use it. Pre-prepare a CTAB solution (10%) by heating and store it at 37C temperature. Add 500 l 70% ethanol, resuspend pellet by flicking, allow to sit for ~5 min at room temp, then spin and drain again. The extraction is repeated on the aqueous phase until it becomes completely clear, and all DNA is collected. %%EOF Add 1/10 volume Na-acetate (50 l) and 2 vol. Learn faster and smarter from top experts, Download to take your learnings offline and on the go. Nucleic acids research. UYZsOhq9_&`YWJ+lZ^m]QfPZ^&UTj`F,M^Mmh!.8MLAnJ^gM>LXI=uC2s Ck@1o(T|! CTAB method is an organic solvent-based extraction method that has health hazards and harmful properties. Main Outcome Measures: Successful extraction of DNA Results: PCR, RFLP and DNA sequencing gave the expected results in all the DNA samples extracted by all the three methods (boiling, microwave irradiation and the commercial kit). other metals binds magnesium. Afterward, the homogenized sample would be incubated with lysis buffer and protease enzyme for effective lysis of the cell wall. Genomic DNA extraction and amplicon generation. Conclusively, PVP and beta-mercaptoethanol must be there in the plant DNA extraction buffer with the CTAB. EB (Elution buffer): 5 mM Tris-HCl. Increasing the heat and duration of the CTAB step might help with tough cells (eg. Now customize the name of a clipboard to store your clips. 4 0 obj _:)a"P$,4+ 5>15Kzv$& This paper is only a guide for these techniques and we describe. insoluble complex with the polysaccharides. In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. CTAB based extraction buffers are widely used when purifying DNA from plant tissues. Precipitation . Treat the sample with PCI, centrifuge it, and collect the supernatant part. Q TP? Lets see how it works and how to optimize the CTAB buffer for plant DNA extraction.. [*ZeJ3"P|7He ]nBx)rgrRSfh3sVPkYQxs811Bwiw"gML xMCs34BA^Co{Ba[*- 8`EAr%EVCq)5U J)\)`.7 DNA extraction - Wikipedia ^ 0=pLRto6Q&N(Na*Y;"I$1k*wkX 5|AQ>4DWii/:&"E N Isolating plant genomic DNA without liquid nitrogen | SpringerLink Cetrimonium bromide - Wikipedia For DNA, absorbance at A 260 (also called optical density, OD) is converted into DNA concentration by following method: A 260/OD of 1.0 = a concentration of 50 g/ml of double-stranded DNA . These are essential for maintaining and further supporting the rapidly evolving pace, scope, and scale of agricultural R&D. DNA extractions using the NGS protocol with (D) no PVP, (E) 1% PVP, and (F) 4% PVP. Nonetheless, to get success every time in plant DNA extraction, the first thing is to understand the structure and constituent of the plant cell of the species you are using. This protocol is wholly adapted from the Coleman Lab protocols, including notes and comments. But differences in the cell structure and chemical composition of a cell require a different DNA extraction setup. By whitelisting SlideShare on your ad-blocker, you are supporting our community of content creators. CTAB - DNA precipitation DNA is hydrophilic and dissolves in water but not in alcohol To separate the DNA from the cell debris, alcohols and salts are used Organic solvents are used to separate the DNA to an aqueous phase, leaving lipids and proteins in the organic phase. %PDF-1.4 % The Fast ID Genomic DNA Extraction Kit is designed for whole grains, fruits, and vegetables, and the Fast ID Ultra DNA Extraction Kit (Catalog No. It was used to extract material for the Micromonas RCC299 complete genome sequencing project, and the Micromonas RCC472 genome sequencing project. Euphrasia nankotaizanensis (Orobanchaceae) is a rare alpine herb that is endemic to Taiwan. However, too much ethanol is also BADDNA will float out of wells on gel. 13,000 rpm at 4C for 10 min, collect the supernatant to a new eppendorf tube, add 600 L of The DNA-containing sample is added to 3mL of lysis buffer (0.4 M NaCl, 10 mM Tris-HCl pH 8.0 and 2 mM EDTA, pH 8.0), SDS and proteinase K. The mixture is incubated at 55-65 overnight. Opeyemi Olu' Ogedengbe - Quality Control Officer - Harvestfield The procedure can also be easily scaled up tenfold to accommodate 50 ml broths.